The Bir1p (survivin)/Ark 1p (Aurora B)/Pic1p (INCENP) chromosome passenger protein complex is conserved in all eukaryotes, and serves to sense spindle microtubule-kinetochore tension to ensure that chromosomes are faithfully segregated at the onset of anaphase. Using reagents we have generated, we plan to carry out biochemical and functional analysis of the Bir1p/Pic1p/Ark1lp complex and its individual components in S. pombe, and identify mitotic substrates for the Ark1lp kinase. These experiments will provide insights into the functions of the Bir1p/Pic1p/Ark1p complex in fission yeast and how Ark1p/Aurora B phosphorylation of target proteins mediates tension sensing and proper chromosome segregation, and signaling to the cleavage furrow. We have shown that Bsh3p, a birl-46 suppressor, is essential for chromosome segregation. We will investigate Bsh3 function in S. pombe and vertebrates, and its relation to the GINS DNA replication complex, and ascertain whether Bsh3p loads Bir1p and other proteins onto replicating chromosomes, thereby linking replication with chromosome segregation in subsequent mitosis. We will also analyze the mitotic function of Rfp1p, an S. pombe Ark1p-interacting protein that binds SUMOylated proteins, to further our understanding of the functions of SUMOylation in mitosis, and also the structural basis for recognition of SUMO by a SUMO-binding domain. In a second aim, we will investigate the mechanism of activation of the Rad3p checkpoint kinase in response to DNA damage, and the role of chromatin modifications and the checkpoint signaling pathway in the repair of DNA damage in S. pombe. For this purpose we will determine whether S. pombe Rad3p, an ATR kinase orthologue, exists as an inactive dimer that dissociates into active monomers in response to DNA damage, and whether intrinsic kinase activity is required for dissociation. We will analyze what changes in histone modification and chromatin structure occur at sites of DNA damage, using a system we have engineered, where a single double-stranded DNA break (DSB) is induced in the S. pombe genome by the I-Scel endonuclease. We will use this system to determine whether chromatin structure and DNA damage checkpoint activation influence the repair mode of I-Scel-induced DSBs. We anticipate that these experiments will provide insights into the mechanism of activation of Rad3p by DNA damage, and an understanding of how chromatin modification at the site of DNA damage is involved in the recognition of DNA damage and how activation of checkpoint pathways by DNA damage can influence the repair processes that are used to repair the damage.